Effects of heavy ions on inactivation and DNA double strand breaks in Deinococcus radiodurans R1.

Inactivation and double strand break (dsb) induction after heavy ion irradiation were studied in stationary phase cells of the highly radiation resistant bacterium Deinococcus radiodurans R1. There is evidence that the radiation sensitivity of this bacterium is nearly independent on energy in the range of up to 15 MeV/u for lighter ions (Ar). The responses to dsb induction for charged particles show direct relationship between increasing radiation dose and residual intact DNA.     

Heavy-ion effects on cellular and subcellular systems: inactivation, chromosome aberrations and strand breaks induced by iron and nickel ions.

A broad spectrum of particles and energies has been used in the last years to study the influence of the radiation quality i.e. of the physical parameters of the particle beam on the biological effectiveness ?2?12?. In these measurements a common structure of the functional dependence of the induction probability per particle (cross section) from the linear energy transfer is observed for different biological endpoints. Because of the relevance for space research, we present in this report our data from experiments with iron and nickel particles, in particular. Our experiments were designed to investigate the relationship between the inactivation and chromosome aberration in mammalian cells and the induction of single and double strand breaks in SV40 DNA in respect to the parameters of the track formation like LET and particle energy.     

Double strand breaks in the DNA of Bacillus subtilis cells irradiated by heavy ions.

Cells of Bacillus subtilis strain TKJ 8431 in stationary phase were irradiated with X-rays (150 kV at DLR) or heavy ions (Ne, Ar, Pb with residual energies between 3 and 15 MeV/u at GSI). The action cross section for the formation of double strand breaks in the DNA of the irradiated cells follows a similar dependence on mass and energy of the ions as has been found for various biological endpoints, e.g. inactivation, mutagenesis and repair efficacy.     

Inactivation, mutation induction and repair in Bacillus subtilis spores irradiated with heavy ions.

Studies on the response of bacterial spores to accelerated heavy ions (HZE particles) help in understanding problems of space radiobiology and exobiology. Layers of spores of Bacillus subtilis strains, differing in repair capabilities, were irradiated with accelerated boron, carbon and neon ions of linear energy transfer (LET) values up to 14000 MeV cm2/g. Inactivation as measured by loss of colony forming ability and induction of mutations as measured by reversion to histidine prototrophy and resistance to 150 micrograms/ml sodium azide were tested, as well as the influence of repair processes on these effects. For inactivation, the cross-sectional values sigma plotted as a function of LET follow a saturation curve. The plateau, which is reached around a LET of 2000 MeV cm2/g, occurs at 2.5 x 10(-9) cm2, a value in good agreement with the dimensions of the spore protoplast. Lethal damage produced at LET values < 2000 MeV cm2/g is reparable. Recombination repair is more effective than excision repair. At higher LET values, lethal damage could not be reconstituted by the repair mechanisms studied. In addition, at these high LET values, the frequency of induced mutations was drastically decreased. The data support the assumption of at least two qualitatively different types of lesion, depending on the LET of the affecting heavy ion.     

Cell inactivation, repair and mutation induction in bacteria after heavy ion exposure: results from experiments at accelerators and in space.

To understand the mechanisms of accelerated heavy ions on biological matter, the responses of spores of B. subtilis to this structured high LET radiation was investigated applying two different approaches. 1) By the use of the Biostack concept, the inactivation probability as a function of radial distance to single particles' trajectory (i.e. impact parameter) was determined in space experiments as well as at accelerators using low fluences of heavy ions. It was found that spores can survive even a central hit and that the effective range of inactivation extends far beyond impact parameters where inactivation by delta-ray dose would be effective. Concerning the space experiment, the inactivation cross section exceeds those from comparable accelerator experiments by roughly a factor of 20. 2) From fluence effect curves, cross sections for inactivation and mutation induction, and the efficiency of repair processes were determined. They are influenced by the ions characteristics in a complex manner. According to dependence on LET, at least 3 LET ranges can be differentiated: A low LET range (app. < 200 keV/micrometers), where cross sections for inactivation and mutation induction follow a common curve for different ions and where repair processes are effective; an intermediate LET range of the so-called saturation cross section with negligible mutagenic and repair efficiency; and a high LET range (>1000 keV/micrometers) where the biological endpoints are majorly dependent on atomic mass and energy of the ion under consideration.     

In vivo and in vitro measurements of complex-type chromosomal exchanges induced by heavy ions.

Heavy ions are more efficient in producing complex-type chromosome exchanges than sparsely ionizing radiation, and this can potentially be used as a biomarker of radiation quality. We measured the induction of complex-type chromosomal aberrations in human peripheral blood lymphocytes exposed in vitro to accelerated H-, He-, C-, Ar-, Fe- and Au-ions in the LET range of approximately 0.4-1400 keV/micrometers. Chromosomes were analyzed either at the first post-irradiation mitosis, or in interphase, following premature condensation by phosphatase inhibitors. Selected chromosomes were then visualized after FISH-painting. The dose-response curve for the induction of complex-type exchanges by heavy ions was linear in the dose-range 0.2-1.5 Gy, while gamma-rays did not produce a significant increase in the yield of complex rearrangements in this dose range. The yield of complex aberrations after 1 Gy of heavy ions increased up to an LET around 100 keV/micrometers, and then declined at higher LET values. When mitotic cells were analyzed, the frequency of complex rearrangements after 1 Gy was about 10 times higher for Ar- or Fe- ions (the most effective ions, with LET around 100 keV/micrometers) than for 250 MeV protons, and values were about 35 times higher in prematurely condensed chromosomes. These results suggest that complex rearrangements may be detected in astronauts' blood lymphocytes after long-term space flight, because crews are exposed to HZE particles from galactic cosmic radiation. However, in a cytogenetic study of ten astronauts after long-term missions on the Mir or International Space Station, we found a very low frequency of complex rearrangements, and a significant post-flight increase was detected in only one out of the ten crewmembers. It appears that the use of complex-type exchanges as biomarker of radiation quality in vivo after low-dose chronic exposure in mixed radiation fields is hampered by statistical uncertainties.     

Quantitative interpretation of heavy ions effects: models for the biological effects of heavy ions.

Heavy ions are an important part of space radiation. Although they contribute only about 1 percent in number the fraction in terms of energy deposited is much higher. Also the quality of radiation is different from the other components since the LET is generally quite high. This poses the problem of Relative Biological Effectiveness (RBE). It is considerably more important in space than on earth because shielding measures are costly and sometimes not even feasible. Radiation hazards appear to be the limiting factor In long term space flights and their evaluation constitutes a major task. There is still no general agreement about RBE of earthbound radiation, and even less concerning the biological weighting of very heavy and very energetic ions in space. Because of the lack of experimental data--particularly for risk estimates in humans-- theoretical approaches may be very helpful in this respect and provide the only means to judge the radiation protection situation in outer space. In order to be useful careful checks of their consistency are necessary. This paper summarizes some of the more common approaches in a critical manner. The unhappy conclusion at the end will be that at present it is not possible to understand even heavy ion action on survival quantitatively with an acceptable precision.     

Biophysical effect of cosmic heavy ions of distinct LET-classes in a plant model system.

Results presented from recent space flight BION 9 show biological effects of different LET-classes of HZE-particles in different target regions of the seed (meristem and the whole embryo) of Arabidopsis thaliana (L.) Heyhn. HZE-one hit events and non-hit events, i.e. only hit by the low-LET background radiation, and their combined effects on the biological damage endpoint lethality are distinguished. This procedure is opening the opportunity of an approach to comparative studies of the biological effects induced by cosmic HZE-particles of different LET-ranges interacting in the complex cosmic radiation spectrum and with other space flight conditions.     

LET dependence of cell death, mutation induction and chromatin damage in human cells irradiated with accelerated carbon ions.

We investigated the LET dependence of cell death, mutation induction and chromatin break induction in human embryo (HE) cells irradiated by accelerated carbon-ion beams. The results showed that cell death, mutation induction and induction of non-rejoining chromatin breaks detected by the premature chromosome condensation (PCC) technique had the same LET dependence. Carbon ions of 110 to 124keV/micrometer were the most effective at all endpoints. However, the number of initially induced chromatin breaks was independent of LET. About 10 to 15 chromatin breaks per Gy per cell were induced in the LET range of 22 to 230 keV/micrometer. The deletion pattern of exons in the HPRT locus, analyzed by the polymerase chain reaction (PCR), was LET-specific. Almost all of the mutants induced by 124 keV/micrometer beams showed deletion of the entire gene, while all mutants induced by 230keV/micrometer carbon-ion beams showed no deletion. These results suggest that the difference in the density distribution of carbon-ion track and secondary electron with various LET is responsible for the LET dependency of biological effects.     

Effect of track structure and radioprotectors on the induction of oncogenic transformation in murine fibroblasts by heavy ions

The oncogenic potential of high-energy ⁵⁶Fe particles (1 GeV/nucleon) accelerated with the Alternating Gradient Synchrotron at the Brookhaven National Laboratory was examined utilizing the mouse C3H 10T1/2 cell model. The dose-averaged LET for high-energy ⁵⁶Fe is estimated to be 143 keV/μm with the exposure conditions used in this study. For ⁵⁶Fe ions, the maximum relative biological effectiveness (RBE_max) values for cell survival and oncogenic transformation were 7.71 and 16.5 respectively. Compared to 150 keV/μm ⁴He nuclei, high-energy ⁵⁶Fe nuclei were significantly less effective in cell killing and oncogenic induction. The prostaglandin E₁ analog misoprostol, an effective oncoprotector of C3H 10T1/2 cells exposed to X rays, was evaluated for its potential as a radioprotector of oncogenic transformation with high-energy ⁵⁶Fe. Exposure of cells to misoprostol did not alter ⁵⁶Fe cytotoxicity or the rate of ⁵⁶Fe-induced oncogenic transformation.     

Effects of heavy ions on visual function and electrophysiology of rodents: the ALTEA-MICE project.

ALTEA-MICE will supplement the ALTEA project on astronauts and provide information on the functional visual impairment possibly induced by heavy ions during prolonged operations in microgravity. Goals of ALTEA-MICE are: (1) to investigate the effects of heavy ions on the visual system of normal and mutant mice with retinal defects; (2) to define reliable experimental conditions for space research; and (3) to develop animal models to study the physiological consequences of space travels on humans. Remotely controlled mouse setup, applied electrophysiological recording methods, remote particle monitoring, and experimental procedures were developed and tested. The project has proved feasible under laboratory-controlled conditions comparable in important aspects to those of astronauts' exposure to particle in space. Experiments are performed at the Brookhaven National Laboratories [BNL] (Upton, NY, USA) and the Gesellschaft für Schwerionenforschung mbH [GSI]/Biophysik (Darmstadt, FRG) to identify possible electrophysiological changes and/or activation of protective mechanisms in response to pulsed radiation. Offline data analyses are in progress and observations are still anecdotal. Electrophysiological changes after pulsed radiation are within the limits of spontaneous variability under anesthesia, with only indirect evidence of possible retinal/cortical responses. Immunostaining showed changes (e.g. increased expression of FGF2 protein in the outer nuclear layer) suggesting a retinal stress reaction to high-energy particles of potential relevance in space.     

Quantitative interpretation of heavy ion effects: comparison of different systems and endpoints.

For a quantitative interpretation of biological heavy ion action the following parameters have to be taken into account: variations of energy depositions in microscopical sites, the dependence of primary lesion formation on local energy density and changes in reparability. They can be studied in objects of different size and with different sensitivities. Results on survival and mutation induction in yeast and in mammalian cells will be compared with theoretical predictions. It is shown that shouldered survival curves of diploid yeast can be adequately described if the final slope is adjusted according to the varying production of primary lesions. This is not the case for mammalian cells where the experiments show a rapid loss of the shoulder with LET, contrary to theoretical expectations. This behaviour is interpreted to mean that the reparability of heavy ion lesions is different in the two systems. Mutation induction is theoretically expected to decrease with higher LET. This is found in yeast but not in mammalian cells where it actually increases. These results suggest a higher rate of misrepair in mammalian cells.     

Physical events in the track structure of heavy ions and their relation to alterations of biomolecules.

Heavy charged particles interacting with biological cells can produce a wide variety of different physical, chemical and biological consequences. A rigorous identification of relevant chemical and biological alterations of biomolecules in cells, however, is still lacking and, thus, it is difficult to identify the potential biological importance of different early physical events. In addition, due to experimental and theoretical problems also little is known about the details of energy transfer, -absorption and -decay from projectiles to atoms/molecules in condensed targets; this is particularly true for not completely stripped heavy ions. Nevertheless, one might conclude from available data that higher densities of physical energy absorption events have a significantly higher probability to lead to qualitatively more severe biochemical alterations as regards the induction of DNA double strand breaks and of chromatin damage. It is not very likely that energy migration along the DNA molecule in biological cells over long distances plays a significant role as contributor to these biological radiation effects.     

The effect of track structure on cell inactivation and chromosome damage at a constant LET of 120 keV/micrometer.

The influence of track structure on chromosome damage and cell inactivation are being investigated. Plateau-phase normal human fibroblast cultures were irradiated with gamma rays, and He, Ne and Ar ions. Particle velocities were chosen so that all beams had an LET of 120 keV/micrometer. In this constant-LET experimental design, the radial distribution of excitations and ionizations about the particle track is the most significant variable. Using premature chromosome condensation, chromatin breaks were measured at two time points, promptly after irradiation and after a prolonged incubation to allow for repair. These measurements give an indication of both initial chromosomal damage and also residual damage that is either not repaired or is misrepaired. Survival was measured under the same conditions. Results indicate that the RBEs for both cell inactivation and, to a lesser extent, chromosome damage decrease as particle energy increases.     

Damage to the photoreceptor cells of the rabbit retina from 56Fe ions: effect of age at exposure, 1.

Optic and proximate tissues of New Zealand white (NZW) rabbits at ages (approximately 3.5 years) near the middle of their median lifespan (5-7 years) were given 0.5-3.5 Gy of 465 MeV u-1 56Fe ions in the Bragg plateau region of energy deposition at a linear energy transfer (LET infinity) of 220 +/- 31 keV micrometer-1. Dose-dependent losses of retinal photoreceptor cells (rods) occurred until 1-2 years after irradiation, the period of this interim report. Similar cumulative losses of photoreceptor cells were seen during the period 1-2 years post-irradiation for rabbits given comparable exposures when young (6-9 weeks old). Since losses of photoreceptor cells at early times had not been determined previously, the current experiment, which was designed to simulate the responses of mature astronauts, redressed that deficiency.     

Preliminary results of space experiment: Implications for the effects of space radiation and microgravity on survival and mutation induction in human cells

In view of the concern for the health of astronauts that may one day journey to Mars or the Moon, we investigated the effect that space radiation and microgravity might have on DNA damage and repair. We sent frozen human lymphoblastoid TK6 cells to the International Space Station where they were maintained under frozen conditions during a 134-day mission (14 November 2008 to 28 March 2009) except for an incubation period of 8 days under 1G or μG conditions in a CO₂ incubator. The incubation period started after 100 days during which the cells had been exposed to 54 mSv of space radiation. The incubated cells were then refrozen, returned to Earth, and compared to ground control samples for the determination of the influence of microgravity on cell survival and mutation induction. The results for both varied from experiment to experiment, yielding a large SD, but the μG sample results differed significantly from the 1G sample results for each of 2 experiments, with the mean ratio of μG to 1G being 0.55 for the concentration of viable cells and 0.59 for the fraction of thymidine kinase deficient (TK⁻) mutants. Among the mutants, non-loss of zygosity events (point mutations) were less frequent (31%) after μG incubation than after 1G incubation, which might be explained by the influence of μG on cellular metabolic or physiological function. Additional experiments are needed to clarify the effect of μG interferes on DNA repair.     

Significant proton events of solar cycle 22 and a comparison with events of previous solar cycles.

The sun has produced several high energy and large fluence solar proton events during solar cycle 22. This recent activity is similar to activity that occurred in the 19th solar cycle before the advent of routine space measurements. In a review of the recent events and a comparison with significant solar proton events of previous solar cycles, it appears that the 20th and 21st solar cycles were deficient in the total fluence of solar particles as detected at the earth. Therefore, when models of maximum solar proton fluxes to be encountered for deep space missions are developed, solar proton data acquired during the present solar cycle should be incorporated.     

Long term effects of low doses of 56Fe ions on the brain and retina of the mouse: ultrastructural and behavioral studies.

Eight month old male C57BL6 mice were exposed without anesthesia to whole-body irradiation in circular holders. The mice were tested for behavioral decrements after 0.5 and 50 rads of Fe particle irradiation at 6 and 12 months post irradiation to obtain long term results. A standard maze was used and the animals were timed for completion thereof. A string test also was administered to the mice, testing their ability to grasp and move along a string to safety. The results from animals exposed to 50 rads were significantly different from [correction of fron] control results to p = < .001 in both systems of testing. The hippocampus (believed to be the location of environmental interaction in the brain) and the retina were examined for ultrastructural changes. The ultrastructural changes were similar to those we found in our Cosmos 782, 936 and in our Argon experiments. The mouse data indicate that iron particles were able to induce long term changes in the central nervous system which lead to behavioral impairment.